Method for the cultivation of microorganisms from body fluid

ABSTRACT

An improved apparatus and method for the collection, cultivation and identification of microorganisms obtained from body fluids is disclosed. The apparatus includes an evacuated tube containing a culture medium, an inert gaseous atmosphere and a vent-cap assembly. The tube containing the culture medium is fitted with a stopper for introduction of body fluid by means of a cannula and after growth of the organisms, transfer of the cultured medium is completed for sub-culturing or identification procedures.

United States Patent Mehl Sept. 9, 1975 [54] METHOD FOR THE CULTIVATION OF 2,992,974 7/1961 Bclcove et a]. l95/l()3.5 R MICROORGANISMS FROM BODY FLUID 3,545,427 12/1970 Rvan 128/2 F 3,730,170 5/1973 Michael 128/2 F [75] Inventor: Jack Judson Mehl, Landing, NJ.

[73] Assignee: Becton Dickinson and Company Primal-v Examiner-Alvin E. Tanenholtz East Rutherford Attorney, Agent, or Firm-Kane, Dalsimer, Kane, [22] Filed: Feb. 11, 1974 Sullivan and Kurucz [21] Appl. No.: 440,969

Related US. Application Data [57] ABSTRACT [60] Division of Ser. No. 342,086, March 16, 1973, and a Confirmation-impart of 215,730, 9 An improved apparatus and method for the collection, 1972' abandonedcultivation and identification of microorganisms obtained from body fluids is disclosed. The apparatus in- [52] Cl 195/109; 195/1035 R; 195/!26; eludes an evacuated tube containing a culture me- 9 195/127; 128/2 F dium, an inert gaseous atmosphere and a vent-cap as- [51] Int. Cl. C12B 1/00; C12B l/24 sembly The tube Containing the culture medium is fit [58] Field Of Search 195/109, 126, 103.5 R; ted i a Stopper f introduction of y fluid y 128/2 F means of a cannula and after growth of the organisms, transfer of the cultured medium is completed for sub- [56] References Cited culturing or identification procedures.

UNITED STATES PATENTS 2,607,347 8/!952 Kleiner 195/1035 R 6 Claims, 12 Drawing Figures -'fllllll/Ill/ PATENTEBSEP ems 4 m 2 W l// 6n B 2 f IWIIIIIIIIII.

SHEET 1 BF {1 PATENTED 91975 sum u 0r {1 METHOD FOR THE CULTIVATION OF MICROORGANISMS FROM BODY FLUID CROSS REFERENCE TO RELATED APPLICATION This application is a division of Ser. No. 342,086 filed Mar. 16, 1973 and is a continuation-in-part of applica tion Ser. No. 215,730 filed on Jan. 6, 1972 now aban doned. 1

BACKGROUND OF THE INVENTION" been necessary to culturethesejum and gaseous atmosphere.

There are three popular techniques for the collection of blood or other body fluids. The collection may be accomplished by the use ofa syringe and needle assembly to withdraw blood from a vein, then inoculation of the blood into two or three sterile containers of media. A second method is to employ an evacuated tube containing an anti-coagulant when collecting blood and subsequently to transfer a portion of the collected blood to the appropriate medium by needle and syringe or by removal of the stopper from the evacuated tube and measuring the appropriate amount of blood specimen by use of a pipette. A third method for the collection of blood employs a piece of flexible tubing having necdle assemblies at each end thereof; one of the needle assemblies adapted for use for entering a vein andthe other for directly inserting into an evacuated tube with or without liquid. and solid culture media disposed tainer which automatically draws a controlled amount of body fluid, particularly blood, so as 'to provide a predetermined ratio of body fluid to medium without the necessity for measuring the quantity of body fluid required. It is alsoan, object of the invention to provide a vent unit assembly adapted to maintain sterility of the stopper after it hasbeen sterilized and prevent contamination of the culture medium during incubation. Also,

-an outlet means is provided forgases generated by the growth .of microorganisms and for theinflow of oxygen to sustain the growth of the aerobic microorganisms. The. outlet and inflow means includes a total path length and a plurality of passages of such small cross section that they constitute a microorganism trap or barrier. It is also an object of the invention to provide a disposable system for collecting and culturing microorganisms, which system is inexpensive to manufacture and simple to use and maintain in aseptic condition when collecting or transferring body fluidsto the culture medium. 7

It is an object of the invention to provide an assembly to simultaneously culture aerobic and anaerobic microorganisms in which the assembly includes means for controlling and limiting the rate of oxygen diffusion through the culture medium. v I

In general, my invention provides an improved system for the collection of 'a specimen directly into a tube containing a medium suitable for the growth of microorganisms contained in the specimen. Various media may be used, as preferred, including agents which change color. thereby identifying certain classes of microorganisms. The system comprises means for automatically collecting the proper proportion of sample so that a predetermined saniple-to-medium ratio is obtained. Means are also provided to effect a'transfer of the sample to the medium under aseptic conditions without special equipment or procedures being employed. Means for maintaining a proper atmosphere within the sample tube for the growth of either aerobic or anaerobic microorganisms in a single culturing contherein. Hox to clamp the tube to prevent additional blood fr iifl is pmvidedentering the container so as to maintain the proper blood-to-medium ratio. It also requires the use of two types of evacuated tubes, one for aerobieand one for anaerobic organisms.

One of the problems encountered while culturing anaerobic microorganisms is the possibility of the stopper being forced from the evacuated tube by the internal gas pressure and, in some instances, where a screw cap is used and later is removed. the stopper may explode from the opening thereby causing contamination of the work area.

In all of the present methods employed strict aseptic techniques must be maintained to avoid foreign bacteria contaminating the sample to be cultured.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an improved culturing system for the collection, cultivation and identification of microorganisms obtained from body fluids. It is another object of the invention to provide a method and apparatus for the cultivation of either aerobic or anaerobic microorganisms or both simultaneously in a single. culturing assembly. A further object of the invention is to provide a collection con- My improved method employs a venipuncture assem bly for collecting blood from a vein such as an assembly employing an evacuated tube and a'holder or barrel fitted with a sample needle. Such an assembly is disclosed in U.S. Pat. No. 2,450,641 and the multiple sample needle is disclosed in U.S. Pat. Nos. 3,469,572 and 3,494,352. An evacuated tube containing a desired culture medium is employed to draw the blood through the sample needle. The evacuated tube is provided with a sufficiently reduced pressure-to collect a predeterminedamount of blood therein and is also provided with an inert atmosphere such as carbon dioxide or nitrogen. The tube stopper is sterilized and then a venting assembly is mounted on the collection tube so as to maintain a sterile field around the stopper during incubation. .The venting unit comprises a barrel portion having a diameter slightly larger than the sample tube stopper and a needle is mounted in the closed end of the barrel. One pointed end of the needle extends into the barrel portion a distance sufficient to penetrate the stopperso as to provide communication between the interior of the tube and the'atmosphere. The other end of the needle extends away from the stopper and is fitted with'a venting. shield including minute passageways so that atmospheric air can enter the culture tube while gases generated from the growth of'microorganisms tom of the culture tube, while facultative and actively motile organisms may be found throughout the culture medium.

Samples of the culture medium are removed from the culture tube employing aseptic techniques to avoid outside contamination. Various procedures may be employed for removal of culture medium. The stopper may be removed together with the venting unit intact. Then, asample from the topmost portion of the culture medium is collected either by use of a sterile pipette or a sterile syringe and needle. The sample is transferred to a sub-culture assembly such as a Petri dish containing theproper solid culture medium for identification of the suspected aerobic organisms. I

A second sample may be taken from the culture tube by insertion of a sterile pipette or sterile needle and syringe assembly so that the tip thereof is disposed near the bottom of the tube. The sample to be cultured is transferred to a sub-culture assembly as before. A third sample may be taken from the middle of the culture by the technique described above. The sub-cultures are cultured in the proper thermal environment and in the proper gaseous atmosphere. aerobic in the presence of DESCRIPTION OF THE DRAWING FIG. 1 is an exploded view in elevation of the culturing assembly of the invention ready for use.

FIG. 2 is a fragmentary view of the venting unit mounted in position on the culturing tube shown in FIG. 1.

,FIG. 3 is an enlarged fragmentary sectional view taken along the lines 33 of FIG. 2.

I FIG. 4 is a sectional view in elevation of the venting unit and the stopper removed from the culture tube of FIG. 2;

FIG. 5 is an elevational view of the culture tube in a holder shown in situ on a patients arm and ready to receive a predetermined volume of blood.

' FIG. 6 is an elevational view showing the culture tube being removed from the holder in situ after the sample of blood is obtained.

FIGS. 7 through 9 illustrate various procedures for removing culture samples from the culture tube.

FIG. 10 is a side elevation showing how gases generated by the microorganisms being cultured may be conducted to a means for analyzing these gases.

FIG. I! is an enlarged fragmentary sectional view in elevation of the venting unit of FIG. 4 having removable plug means mounted in the cannula.

FIG. 12 is a side elevation, partly in section, of another form of the container for the culture medium, with the venting unit mounted in place.

DESCRIPTION OF THE PREFERRED EMBODIMENT The improved culture system for culturing microorganisms either strictly aerobic or anaerobic is shown in FIG. I in exploded detail and is referred to by the numeral 10. An evacuated tube or container 12 is employed which may be formed of glass or a transparent plastic material, and is non-toxic to and inert to the cul ture medium and organisms to be cultured. A stopper 14'is removably mounted in the open end 15 of the culture tube 12.

A venting unit 16 is mounted on tube 12 as illustrated in FIG. 2. The venting unit includes a body portion 18 which is made of a flexible plastic, for example, polyethylene; The body portion 18 is cylindrically shaped having a diameter slightly larger than stopper 14 of culture tube 12 and is open at one end so that the stoppered end of the tube will be capped by venting unit 16 as shown in FIG. 2. The other end of the body portion is closed by an end wall l9. A hub or tip 20 is formed at the center of wall 19 and has an axial bore 21 formed therethrough so that cannula 24 will be positioned axially of body portion 18. Cannula 24 is rigidly mounted in'bore 21 by anysuitable means, such by epoxy. Cannula 24 is mounted in tip 20 between ends 25 and 26. End 25 is pointed to facilitate piercing stopper 24 so that fluid communication is maintained between the interior of tube 12 and the atmosphere.

As shown in FIG. 2 the venting unit is mounted in position with the pointed end 25 of the cannula 24 extending through the stopper 14 so that the pointed end 25 is spaced from the top of the culture medium 30. When the assembly 10 is placed in the proper thermal environment an exchange of atmosphere is accomplished by cannula 24 by passing gases contained in the space above the culture medium through cannula 24 to the atmosphere and passing the proper atmosphere into the space above the culture media.

Venting unit 16 also includes a plastic shield 28 which is formed having internal ridges 29, as shown in FIG. 3, disposed around the interior thereof so that when it is removably mounted on top 20, the ridges contact the outer surface of tip 20 to form passageways 31 for conducting the atmosphere contained within the culture unit through the cannula 24 and subsequently may be expelled to the outside atmosphere. At the same time, the outside atmosphere will pass through the passageways 31 between ridges 29, and through cannula 24, so that the aerobic microorganisms contained in the culture unit will grow at an optimum rate.

In FIG. 11 which illustrates another form of the venting unit, the passageway through cannula 24 may be plugged initially with a viscous material 52 such as a mixture of petroleum jelly and paraffin. This may be used when one desires that all oxygen be excluded from the medium initially when culturing anaerobic microorganisms. Then, as the gas pressure generated by these microorganisms, for example, CO methane, or gas other than oxygen increases, the plug 52 of viscous ma= terial is pushed out of cannula 24; thereby establishing an open continuous passageway from the surface of the medium tothe outside atmosphere, and thereby eliminating any possibility of gas pressure blowing out the stopper. At this advanced stageof growth, diffusion of atmospheric oxygen through the passageways to the surface of the medium would have no deleteriousor inhibiting effect on the growth of the anaerobes.

In FIG. 4 the venting unit 16 is-depicted illustrating one of the techniques employed to remove stopper 14 from culture tube 'l2 to obtain-a sample of culture me. dium while maintaining aseptic conditions. Body l8 of the venting unit 16 is squeezed and by pulling upwardly the stopper is removed from tube. l2.. Thus,1 as illustrated,.stopper 14 is shown piercedbycannula 24 with the pointed bevelled end 25 extending therethrough.

FIG. 7 illustrates the culture tube 12 with stopper l4 removed. A pipette'40 fitted with a resilient bulb 42 at its outer end, is disposed into thetube so that the lower pointed end of the pipette is immersed in the culture medium. Any desired amount of inoculated culture medium may be removed for sub-culturing purposes.

FIG.,8 illustrates still another method of removing a sample from culture tube 12 for sub-culturing purposes. The venting unit 16 isremoved and a sterile syringe 44 and needle assembly isused to withdraw culture medium from tube 12 by piercing stopper 14 with the needle 45. The needle is of sufficient length so thatits pointed bevelled end 46 is immersed in the culture medium;

In FIG. 9 still another method for remove] of culture medium 30 is illustrated. A micro pipette havingv cylindrical body 47 with a diameter sufficient to be slidably disposed within cannula 24 of the venting unit 16. is employed. The micro pipette is illustrated having a flexible bulb 48 mounted on the cylindrical-body. It should be understood that any other means for withdrawing the sample may be employed, for example a hypodermic syringe with a long needle inserted through the cannula 24. After the sample is removed. the shield 28 is replaced over tip 20 to maintain aseptic conditions.

FIG. 10 shows another use of the invention herein. The venting unit 16 is shown with the shield. 28 removed. A flexible tube 50 is coupled to the outer end 26 of cannula 24 so that the gases contained within the culturetube 12 will be conducted through flexible'tube 50 and analyzed by any suitable means so that the presence of a gas peculiar to certain types of organisms can be detected. Also, the volume of gas being generated by the organisms will be indicative of the growth rate of the culture within the tube.

In the embodiment illustrated in FIG. 12 container 55 may be shaped approximately as shown. Tubular neck portion 54 is approximately the size and shape of the upper portion of container 12, so as to cooperate with the venting unit 16, in the manner as previously described and shown in FIG. 4. The primary advantage for having the container 55 in the form illustrated in FIG. 12 is to provide for a much larger blood sample. If only a few of a given type of microorganism are present in the blood stream, statistically thereis amuch better chance to detecting this type of microorganism if the size of the blood sample can be substantially increased while maintaining tlie 1 to Sto 1 to 10 dilution ratio for culturing. 7

Another-advantage for having container 55 as illustrated in FIG. 12 is to provide a base which will permit container 55 to stand upright without having to be placed in a tube rack or holder.

1 It is contemplated when employing the invention herein that any suitable culture medium may be used to culture microorganisms that maybe contained in body fluids. An example of a culturing medium that may be used is Supplemented Peptone Broth blood culture medium, sold'by Becton, Dickinson and Company. Many culture mediums .are available commercially for culturing specific microorganismsand many are modified for culturing several types of microorganisms. The body fluids-that may be employed for culturing purposes when using the invention of the assembly herein are, for example, synovial fluid, spinal fluid, peritoneal fluid, pleural fluid; urine,'blood,-etc- The reason the very desirable culturing of both aerobic and anaerobic microorganisms simultaneously is possible-with the assembly of the invention herein is that design factors in combination are utilized providing-a very largediminishing oxygen gradient. Even though oxygen at nor-ma] atmospheric-partial pressure is present outside the device of the invention, the design factorsthereof are so proportioned and combined that the amount of oxygen diffusing toward the bottom of the column vof the culture medium is less than that which willadversely affect the growth-and multiplication of'anaerobic .microorganisms contained therein. The-significant design factors are the following stated in ,terms of diminishing the rate of oxygen diffusion in respect to:

l. the total cross section of the microbial trap, i.e. passageways 31 formed between ribs 29, the smaller the cross section the less the diffusion rate;

-2. the longer the passageway 31 ofthe microbial trap the smaller the diffusion;

'3. the smaller thecross sectional diameter of the lumen of thecannula 24 of the venting unit 16, the smaller the oxygen diffusion; v

- 4. the longer the lumen of the cannula 24 of the venting unit 16, the smallerthe oxygen diffusion;

5. the smaller the cross sectional area of the top of thecolumn of culture medium 30 the smaller the oxygen diffusion; and i I ,6. the longer the column of culture'medium 30 (from top to bottom).the smaller the oxygen diffusion.

The ranges of dimensions of these factors in combination, in .a preferred form of the invention, are substantially as follows;

1. the total cross sectional area of the passageways 31 of the microbial trap ranging from 0.000025 square inch to 0.002500 square. inch in combination with:

2. the length of the passageways 31 of the microbial trap ranging from 0.200 inches to 0.625 inches, in combination with;

3. the diameter of the lumen of the cannula of the venting unit ranging from 0.016 to 0.052 inches, in combination with;

4. the length of the lumen of the cannula of the venting unit ranging from 0.50 to 2.000 inches,-in combination with; u

5. the total cross sectional area of the top of the colurnn of culture medium equivalent to a disc ranging from a diameter of l0millimeters to 20 millimeters, in combination with;

6. a length of culture medium column ranging from 50 millimeters to 200 millimeters.

The following example is illustrative of the use of the invention herein for culturing microorganisms which may be either strict anaerobic or strict aerobes in the same culturing tube employing the same culturing medium where the body fluid is blood.

EXAMPLE A blood collecting assembly is employed, such as is disclosed in U.S. Pat. Nos. 3,469,572 and 3,494,352. where a culturing specimen is to be collected through a multiple sample needle along with other blood speci mens for hematology or other determinations in the laboratory, utilizing the same venipuncture. Where only a culture specimen is required the blood collecting assembly may be employed such as is disclosed in U.S. Pat. No. 2,460,641. The blood collecting assembly of these patents includes a blood sampling needle a holder H for mounting the needle therein and for receiving and directing an evacuated tube into the assembly with the needle. Initially a venipuncture is made in the usual manner. A tube 16 mm in diameter containing a predetermined volume of gaseous carbon dioxide and nitrogen maintained at a predetermined pressure less than atmospheric pressure and containing a liquid blood culturing medium the length of which is 120 mm is placed in the holder The culturing medium used is Supplemented Peptone Broth blood culturing medium sold by Becton. Dickinson and Company. As illustrated in FIG. 5. the tube 12 is moved into the holder H so that the stopper 14 is penetrated by one end of the blood sampling needle Only a predetermined amount of blood will enter the tube so that the bloodto-culture medium ratio ranges from between 1 to 8 to l to 10. The culturing tube is withdrawn from the holder H" as illustrated in FIG. 6 and is ready to be sent to the laboratory. Before placing the tube in the proper thermal environment the contents are thoroughly mixed by repeated inversion and a venting unit is mounted on the culturing tube such that the stopper is punctured by a venting cannula mounted in the venting unit. The venting cannula has a length of 1.45 inches and a lumen diameter of 0.040 inches. The venting unit is fitted with a venting cap to maintain sterility and to provide a flow of gaseous atmosphere into and microbial gaseous byproducts out of the culture tube. The venting cap provides a plurality of passageways forming a microbial trap so that the diffusion of gaseous atmosphere into the culture tube is maintained at a controlled rate. The passageways have a cross sec tional area of 0.00025 square inches and a length of 0.625 inches. The rubber stopper of the culturing tube is maintained in aseptic condition to prevent outside contamination by bacteria. Where aerobic or both anaerobic and aerobic culturing is to take place, the atmosphere within the incubator is air. or air plus added carbon dioxide.

The culturing assembly is maintained in an upright position in an incubator in the proper thermal environment and atmosphere for a predetermined period such as for 12 to 24 hours. A sample is then removed for sub-culturing or staining. Any of the techniques for removing specimens from the culturing tube for subculturing as set forth in FIGS. 7 through 9 may be employed.

Also. another method for removing a sample of incubated culture medium and blood is to remove the venting unit; mix the cultured medium and blood sample by repeated inversion; replace the venting unit; remove the shield 28; and invert the container so that the venting unit is downward and shake gently to expel one or more drops of liquid through cannula 24 onto a receiving surface. such as a sub-culture medium or slide. It should be remembered, however, that any of the procedures followed must be under aseptic conditions to prevent outside contamination.

lt is obvious where body fluids other than blood are to be employed the culturing medium may be modified accordingly particularly where a specific type of organism may be suspected of being present. Many modifications and changes may be made without departing from the teachings herein and as defined in the appended claims.

I claim:

1. The method of culturing microorganisms obtained from a specimen of body fluid, which comprises:

a. providing an apparatus which comprises,

i. a container having at least one closed end and an open end;

ii. resilient closure means closing said open end;

iii. a nutrient medium partially filling said container and which is suitable for culturing microorganisms contained in said specimen;

iv. a gaseous atmosphere filling the space above said nutrient medium; and

v. a venting assembly mounted on the container and having means for maintaining fluid communication between the outside atmosphere and the atmosphere within the container. said venting assembly comprising a tubular member passing through said resilient closure to provide fluid communication between said gaseous atmosphere above the nutrient medium and the atmosphere outside of said container. said venting assembly including a flexible body portion having a closed end and an open end so that the closure means fitted in the open end of the container is capped thereby; the closed end of the body portion being formed with a hub having an axial bore therethrough and said tubular member is a cannula rigidly mounted in the axial bore between the ends of said bore and wherein a shield is mounted on the hub to enclose the portion of the cannula extending outwardly from the closed end of the body portion, the shield being formed with a plurality of ribs which are radially disposed around the inner surface thereof so that the shield when mounted on the hub forms a plurality of passageways for the circulation of microbial gases from the interior of the container to the outside atmosphere and for diffusion of atmospheric oxygen from the outside to the inside of the container, said passageways being of such small cross section as to constitute a microbial barrier;

b. inoculating said culture medium disposed in said container with a specimen of body fluid to be cultured; and l incubating said inoculated culture medium under environmental conditions of temperature. gaseous atmosphere and for a requisite period of time to facilitate maximum growth of any organisms present in said inoculated culture, whereby simultaneously any aerobic microorganisms will grow and multiple in the top portion of the medium and any anaerobic microorganisms will simultaneously grow and multiply in the bottom portion of the culture medium while any facultative and actively motile microorganisms will grow and multiply throughout the medium.

2. The method of claim 1 wherein said gaseous atmosphere is non-oxidizing.

3. The method of claim 1 including the step of transferring a portion of said inoculated medium for subculturing to identify said microorganisms being cultured.

4. The method of claim 1 wherein the specimen being cultured is blood.

5. A method of culturing microorganisms from a specimen of body fluid which comprises:

a. providing an apparatus which comprises,

i. a container having at least one closed end and an open end;

ii. resilient closure means closing said open end;

iii. a nutrient medium partially filling said container and which is suitable for culturing microorganisms contained in said specimen;

iv. a gaseous atmosphere filling the space above said nutrient medium; and

v. a venting assembly mounted on the container and having means for maintaining fluid communication between the outside atmosphere and the atmosphere within the container; said venting assembly comprising a tubular member passing through said resilient closure to provide fluid communication between said gaseous atmosphere above the nutrient medium and the atmosphere outside of said container, said venting as sembly including a flexible body portion having a closed end and an open end so that the closure means fitted in the open end of the container is capped thereby; the closed end of the body portion being formed with a hub having an axial bore therethrough and said tubular member is a cannula rigidly mounted in the axial bore between the ends of said bore wherein a shield is mounted on the hub to enclose the portion of the cannula extending outwardly from the closed end of the body portion, the shield being formed with a plurality of ribs which are radially disposed around the inner surface thereof so that the shield when mounted on the hub forms a plurality of passageways for the circulation of microbial gases from the interior of the container to the outside atmosphere and for diffusion of atmospheric oxygen from the outside to the inside of the container, said passageways being of such small cross section as to constitute a microbial barrier; wherein a pressure responsive, gas impermeable barrier is disposed in said cannula, said barrier being removable by a pre-determined increase in gas pressure within said container, generated by any microorganisms being cultured within said container;

b. inoculating said culture medium disposed in said container with a specimen of body fluid to be cultured; and

incubating said inoculated culture medium under environmental conditions of temperature, gaseous atmosphere and for a requisite period of time to facilitate maximum growth of any organisms present in said inoculated culture, whereby simultaneously any aerobic microorganisms will grow and multiply in the top portion of the medium and any anaerobic microorganisms will simultaneously grow and multiply in the bottom portion of the culture medium while any facultative and actively motile microorganisms will grow and multiply throughout the medium.

6. The method of claim 5 wherein the specimen being cultured is blood.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 5,904,482 Dated September 9, 1975 Invent0r(s) aCk Judson Mehl It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, line 22, after the word "culture" delete the the word "thesejum" and insert in its place these in separate containers each having the proper medium Column 1, line #2 after the word "therein." delete the word "Hox" and insert in its place However when using this a technique it is necessary Column 2, line #7, delete "2, +5O,6 +l" and insert in its place 2, I6O,6 l-l Column LI, line 36, after the word "stopper" delete the numeral "ZLI" and insert in its place Signed and Scaled this ninth Of December 1975 '[SEAL] Attest:

q RUTH c. MASON c. MARSHALL DANN A resting Office Commissioner ofPatenrs and Trademarks ORM PO-l 050 (10-69) USCOMM-DC 60376-P69 u.s. GOVEKNMENT PRINTING OFFICE 930 I 

1. THE METHOD OF CULTURING MICROORGANISMS OBTAINED FROM A SPECIMEN OF BODY FLUID, WHICH COMPRISES: A. PROVIDING AN APPARATUS WHICH COMPRISES, I. A CONTAINER HAVING AT LEAST ONE CLOSED END AND AN OPEN END, II, RESILIENT CLOSURE MEANS CLOSING SAID OPEN END, III. A NUTRIENT MEDIUM PARTIALLY FILING SAID CONTAINER AND WHICH IS SUITABLE FOR CULTURING MICROORGANISMS CONTAINED IN SAID SPECIMEN, IV. A GASEOUS ATMOSPHERE FILLING THE SPACE ABOVE SAID NUTRIENT MEDIUM, AND V.A VENTING ASSEMBLY MOUNTED ON THE CONTAINER AND HAVING MEANS FOR MAINTAINING FLUID COMMUNICATION BETWEEN THE OUTSIDE ATMOSPHERE AND THE ATMOSPHERE WITHIN THE CONTAINER, SAID VENTING ASSEMBLY COMPRISING A TUBULAR MEMBER PASSING THROUGH SAID RESILIENT CLOSURE TO PROVIDE FLUID COMMUNICATION BETWEEN SAID GASEOUS ATMOSPHERE ABOVE THE NUTRIENT MEDIUM AND THE ATMOSPHERE OUTSIDE OF SAID CONTAINER, SAID VENTING ASSEMBLY INCLUDING A FLEXIBLE BODY PORTION HAVING A CLOSED END AND AN OPEN END SO THAT THE CLOSURE MEANS FITTED IN THE OPEN END OF THE CONTAINER IS CAPPED THEREBY, THE CLOSED END OF THE BODY PORTION BEING FORMED WITH A HUB HAVING AN AXIAL BORE THERETHROUGH AND SAID TUBULAR MEMBER IS A CANNULA RIGIDLY MOUNTED IN THE AXIAL BORE BETWEEN THE ENDS OF SAID BORE AND WHEREIN A SHIELD IS MOUNTED ON THE HUB TO ENCLOSE THE PORTION OF THE CANNULA EXTENDING OUTWARDLY FROM THE CLOSED END OF THE BODY PORTION, THE SHIELD BEING FORMED WITH A PLURALITY OF RIBS WHICH ARE RADIALLY DISPOSED AROUND THE INNER SURFACE THEREOF SO
 2. The method of claim 1 wherein said gaseous atmosphere is non-oxidizing.
 3. The method of claim 1 including the step of transferring a portion of said inoculated medium for subculturing to identify said microorganisms being cultured.
 4. The method of claim 1 wherein the specimen being cultured is blood.
 5. A method of culturing microorganisms from a specimen of body fluid which comprises: a. providing an apparatus which comprises, i. a container having at least one closed end and an open end; ii. resilient closure means closing said open end; iii. a nutrient medium partially filling said container and which is suitable for culturing microorganisms contained in said specimen; iv. a gaseous atmosphere filling the space above said nutrient medium; and v. a venting assembly mounted on the container and having means for maintaining fluid communication between the outside atmosphere and the atmosphere within the container; said venting assembly comprising a tubular member passing through said resilient closure to provide fluid communication between said gaseous atmosphere above the nutrient medium and the atmosphere outside of said container, said venting assembly including a flexible body portion having a closed end and an open end so that the closure means fitted in the open end of the container is capped thereby; the closed end of the body portion being formed with a hub having an axial bore therethrough and said tubular member is a cannula rigidly mounted in the axial bore between the ends of said bore wherein a shield is mounted on the hub to enclose the portion of the cannula extending outwardly from the closed end of the body portion, the shield being formed with a plurality of ribs which are radially disposed around the inner surface thereof so that the shield when mounted on the hub forms a plurality of passageways for the circulation of microbial gases from the interior of the container to the outside atmosphere and for diffusion of atmospheric oxygen from the outside to the inside of the container, said passageways being of such small cross section as to constitute a microbial barrier; wherein a pressure responsive, gas impermeable barrier is disposed in said cannula, said barrier being removable by a pre-determined increase in gas pressure within said container, generated by any microorganisms being cultured within said container; b. inoculating said culture medium disposed in said container with a specimen of body fluid to be cultured; and incubating said inoculated culture medium under environmental conditions of temperature, gaseous atmosphere and for a requisite period of time to facilitate maximum growth of any organisms present in said inoculated culture, whereby simultaneously any aerobic microorganisms will grow and multiply in the top portion of the medium and any anaerobic microorganisms will simultaneously grow and multiply in the bottom portion of the culture medium while any facultative and actively motile microorganisms will grow and multiply throughout the medium.
 6. The method of claim 5 wherein the specimen being cultured is blood. 